1. Field of the Invention
The present invention relates to methods and devices for assaying proteins in a microfluidic environment.
2. Description of the Related Art
Prior art methods for assaying proteins are generally based on two-dimensional electrophoresis (2DE) and Edman degradation. Unfortunately, current 2DE methods allow only the most abundant proteins to be identified. Thus, most of the proteins identified by 2DE methods represent structural proteins or housekeeping proteins. See e.g. Gygi et al. (2000) PNAS USA 97:9390-9395; Gygi et al. (1999) Electrophoresis 20:310-319; Shevchenko (1996) PNAS USA 93:14440-14445; Boucherie (1996) Electrophoresis 17(11):1683-1699; Ducret (1998) Protein Science 7:706-719; and Garrels (1994) Electrophoresis 15:1466-1486. Thus, 2DE prior art methods are unsuitable for assaying proteins in micro or trace amounts.
The lack of sensitivity of current 2DE-based technology is caused primarily by a lack of separating or resolving power because high abundance proteins mask the identification of low abundance proteins. Loading more protein on the gels does not improve the situation because the Gaussian tails of the high abundance spots contaminate the low abundance proteins. The use of zoom gels (2D gels that focus on a narrow pH range) allows for minimal gains but are considered too cumbersome to be of any practical utility. See Corthals (2000) Electrophoresis 21:1104-1115. Selective enrichment methods also can be used but generally at the expense of obtaining a comprehensive view of cellular protein expression. The sensitivity of detection on 2DE gels also is problematic, because the amount of protein required for identification by mass spectrometry (MS) is near the detection limits of the most sensitive methods for visualization of the protein spots on the 2DE gels. Further, the polyacrylamide matrix typically used in 2DE gives rise to a significant amount of background in the extracted sample mixture making subsequent analysis by MS difficult. See Kinter (2000) In Protein Sequencing and Identification Using Tandem Mass Spectrometry, Wiley, N.Y. Additionally, conventional peptide extractions following in-gel digestion procedures result in substantial protein losses which are significantly detrimental to low abundance proteins. See Timperman (2000) Anal. Chem. 72:4115-4121.
Multi-dimensional column separations offer advantages over 2DE, including a higher separating power and reduced sample contamination and loss. A typical large format 2DE gel is capable of achieving a peak capacity of about 2,000 while 2D column separations can achieve peak capacities of over 20,000 for protein separations. Additionally, the stationary phases of these columns are very stable and non-reactive compared to polyacrylamide gels, leading to reduced sample contamination and loss. Many different types of separation techniques have been coupled to 2D column separations including size exclusion, reversed phase chromatography, cation-exchange chromatography, and capillary electrophoresis. See Wall (2000) Analytical Chemistry 72:1099-1111; Link (1999) Nature Biotechnology 17:676-682; Opiteck (1998) Journal of Microcolumn Separations 10:365-375; Hooker et al. (1998) In High-Performance Capillary Electrophoresis, John Wiley & Sons Inc, New York, Vol. 146, pp 581-612; Opiteck et al. (1998) Analytical Biochemistry 258:349-361; Vissers (1999) Journal of Microcolumn Separations 11:277-286; and Liu et al. (1996) Anal. Chem. 68:3928-3933. Unfortunately, multi-dimensional column separations are impractical for use in or in conjunction with microfluidic applications and devices.
Edman degradation allows protein sequencing and involves the cyclic removal and identification of the terminal amino acid based on a labeling reaction between the terminal amino group and phenylisothiocyanate (PITC). When the labeled protein is treated with acid, the N-terminal amino acid residue is cleaved as an unstable intermediate that undergoes rearrangement to a phenylthiohydantoin. The cleaved product can be identified by comparison with phenylthiohydantoin preparations of standard amino acids. The sequence of the protein or polypeptide is elucidated by cycling the protein through many stages of removal and sequential identification of the terminal amino acid residue. Alternative Edman degradation reactions employ PITC analogues and cleavage and identification of C-terminal residues.
Recently, devices for Edman degradation reactions have been miniaturized. See Wurzel & Brigitte (2000) Proteomics in Functional Genomics 88:145-157; Wurzel & Brigitte (1998) Springer, Berlin, Germany:219-224; Wurzel & Brigitte (1998) J. Protein Chem. 17(6):561-564; and U.S. Patent Application Publication No. 20040175822. Unfortunately, these devices are unsuitable for coupling or multiplexing with devices and methods for microfluidic separations and they do not allow the identification of the protein sequence directly from the microfluidic device.
Thus, a need still exists for a devices and methods for assaying and sequencing micro quantities of proteins.